Standard microbiological methods have relied on substrate-based assays to test for the presence of specific organisms (Bordner, et al. 1978). These techniques offer very high levels of selectivity but are hindered by the requirement to first grow or cultivate pure cultures of the targeted organism, which can take 24 hours or longer. This time constraint severely limits the effectiveness to provide a rapid response to the presence of virulent strains of microorganisms.
Molecular biology techniques are quickly gaining acceptance as valuable alternatives to standard microbiological tests. In particular, serological methods have been widely employed to evaluate a host of matrices for targeted microorganisms (Kingsbury & Falkow 1985; Wyatt et al. 1992). These tests focus on using antibodies to first trap and then separate targeted organisms from other constituents in complicated biological mixtures. Once isolated, the captured organism can be concentrated and detected by a variety of different techniques that do not require cultivating the biological analyte.
One very popular approach, termed immunomagnetic separation (IMS), involves immobilizing antibodies to spherical, micro-sized paramagnetic beads and using the antibody-coated beads to trap targeted microorganisms from liquid media. The beads are easily manipulated under the influence of a magnetic field facilitating the retrieval and concentration of targeted organisms. Moreover, the small size and shape of the beads allow them to become evenly dispersed in the sample, accelerating the rate of interaction between bead and target. These favorable characteristics lead to reductions in assay time and help streamline the analytical procedure making it more applicable for higher sample throughput and automation.
Downstream detection methods previously used with IMS include ELISA (Cudjoe, et al. 1995), dot blot assay (Skjerve et al. 1990), electrochemiluminescence (Yu and Bruno 1996), and flow cytometry (Pyle, et al. 1999). Although these tests provide satisfactory results, they are laborious to perform and give binary responses (yes/no) that are highly susceptible to false-positive results due to cross-reactivity with non-target analytes. Recently reported is a rapid method for identifying specific bacteria from complex biological mixtures using IMS coupled to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS)(Madonna et al. 2001). This approach allowed a variety of matrices to be evaluated for the presence of a Salmonella species within a total analysis time of 1 hour. Moreover, the developed procedure required little sample processing, was relatively easy to perform, and the molecular weight information obtained made it possible to discriminate between signals from the target bacteria and signals from cross-reacted constituents.
MALDI-TOF-MS is a proven technique for identifying whole cellular microorganisms (Holland et al (1996); van Barr 2000; Madonna et al. 2000). In principle, MALDI is a ‘fingerprinting’ technique where mass spectra featuring varying distributions of protein signals are generated. The signature profiles that are produced, due to inherent differences in microbial proteomes, make it possible to discriminate between organisms down to the strain level (Arnold and Reilly 1998). The MALDI-TOF technique coupled with IMS includes, in one embodiment, mixing immunomagnetic beads specific to the target bacteria with the liquid mixture that may contain the target bacteria for a short incubation period (e.g., 20 min). Any target bacteria captured by the beads are washed twice, re-suspended in deionized H2O, and directly applied onto a MALDI sample probe. The target bacteria-bead complex is then overlaid with a micro-volume of matrix solution and dried at room temperature. Irradiation of the resulting crystalline mass with a high intensity laser promotes the liberation and ionization of intact cellular proteins that are subsequently detected by a TOF mass spectrometer. The resulting mass spectrum is then interrogated for definitive mass peaks that signify the presence of the target bacteria.